Biofabrication Strategies for Oral Soft Tissue Regeneration.

Gingival recession, a prevalent condition affecting the gum tissues, is characterized by the exposure of tooth root surfaces due to the displacement of the gingival margin. This review explores conventional treatments, highlighting their limitations and the quest for innovative alternatives. Importantly, it emphasizes the critical considerations in gingival tissue engineering leveraging on cells, biomaterials, and signaling factors. Successful tissue-engineered gingival constructs hinge on strategic choices such as cell sources, scaffold design, mechanical properties, and growth factor delivery. Unveiling advancements in recent biofabrication technologies like 3D bioprinting, electrospinning, and microfluidic organ-on-chip systems, this review elucidates their precise control over cell arrangement, biomaterials, and signaling cues. These technologies empower the recapitulation of microphysiological features, enabling the development of gingival constructs that closely emulate the anatomical, physiological, and functional characteristics of native gingival tissues. The review explores diverse engineering strategies aiming at the biofabrication of realistic tissue-engineered gingival grafts. Further, the parallels between the skin and gingival tissues are highlighted, exploring the potential transfer of biofabrication approaches from skin tissue regeneration to gingival tissue engineering. To conclude, the exploration of innovative biofabrication technologies for gingival tissues and inspiration drawn from skin tissue engineering look forward to a transformative era in regenerative dentistry with improved clinical outcomes.

GelMA/TCP nanocomposite scaffold for vital pulp therapy.

Pulp capping is a necessary procedure for preserving the vitality and health of the dental pulp, playing a crucial role in preventing the need for root canal treatment or tooth extraction. Here, we developed an electrospun gelatin methacryloyl (GelMA) fibrous scaffold incorporating beta-tricalcium phosphate (TCP) particles for pulp capping. A comprehensive morphological, physical-chemical, and mechanical characterization of the engineered fibrous scaffolds was performed. In vitro bioactivity, cell compatibility, and odontogenic differentiation potential of the scaffolds in dental pulp stem cells (DPSCs) were also evaluated. A pre-clinical in vivo model was used to determine the therapeutic role of the GelMA/TCP scaffolds in promoting hard tissue formation. Morphological, chemical, and thermal analyses confirmed effective TCP incorporation in the GelMA nanofibers. The GelMA+20%TCP nanofibrous scaffold exhibited bead-free morphology and suitable mechanical and degradation properties. In vitro, GelMA+20%TCP scaffolds supported apatite-like formation, improved cell spreading, and increased deposition of mineralization nodules. Gene expression analysis revealed upregulation of ALPL, RUNX2, COL1A1, and DMP1 in the presence of TCP-laden scaffolds. In vivo, analyses showed mild inflammatory reaction upon scaffolds' contact while supporting mineralized tissue formation. Although the levels of Nestin and DMP1 proteins did not exceed those associated with the clinical reference treatment (i.e., mineral trioxide aggregate), the GelMA+20%TCP scaffold exhibited comparable levels, thus suggesting the emergence of differentiated odontoblast-like cells capable of dentin matrix secretion. Our innovative GelMA/TCP scaffold represents a simplified and efficient alternative to conventional pulp-capping biomaterials. STATEMENT OF SIGNIFICANCE: Vital pulp therapy (VPT) aims to preserve dental pulp vitality and avoid root canal treatment. Biomaterials that bolster mineralized tissue regeneration with ease of use are still lacking. We successfully engineered gelatin methacryloyl (GelMA) electrospun scaffolds incorporated with beta-tricalcium phosphate (TCP) for VPT. Notably, electrospun GelMA-based scaffolds containing 20% (w/v) of TCP exhibited favorable mechanical properties and degradation, cytocompatibility, and mineralization potential indicated by apatite-like structures in vitro and mineralized tissue deposition in vivo, although not surpassing those associated with the standard of care. Collectively, our innovative GelMA/TCP scaffold represents a simplified alternative to conventional pulp capping materials such as MTA and Biodentine™ since it is a ready-to-use biomaterial, requires no setting time, and is therapeutically effective.

Injectable Methacrylated Gelatin Hydrogel for Safe Sodium Hypochlorite Delivery in Endodontics.

Keeping sodium hypochlorite (NaOCl) within the root canal is challenging in regenerative endodontics. In this study, we developed a drug delivery system using a gelatin methacryloyl (GelMA) hydrogel incorporated with aluminosilicate clay nanotubes (HNTs) loaded with NaOCl. Pure GelMA, pure HNTs, and NaOCl-loaded HNTs carrying varying concentrations were assessed for chemo-mechanical properties, degradability, swelling capacity, cytocompatibility, antimicrobial and antibiofilm activities, and in vivo for inflammatory response and degradation. SEM images revealed consistent pore sizes of 70-80 µm for all samples, irrespective of the HNT and NaOCl concentration, while HNT-loaded hydrogels exhibited rougher surfaces. The hydrogel's compressive modulus remained between 100 and 200 kPa, with no significant variations. All hydrogels demonstrated a 6-7-fold mass increase and complete degradation by the seventh day. Despite an initial decrease in cell viability, all groups recovered to 65-80% compared to the control. Regarding antibacterial and antibiofilm properties, 12.5 HNT(Double) showed the highest inhibition zone on agar plates and the most significant reduction in biofilm compared to other groups. In vivo, the 12.5 HNT(Double) group displayed partial degradation after 21 days, with mild localized inflammatory responses but no tissue necrosis. In conclusion, the HNT-NaOCl-loaded GelMA hydrogel retains the disinfectant properties, providing a safer option for endodontic procedures without harmful potential.

Calcium Trimetaphosphate-Loaded Electrospun Poly(Ester Urea) Nanofibers for Periodontal Tissue Engineering.

The objective of this research was to create and appraise biodegradable polymer-based nanofibers containing distinct concentrations of calcium trimetaphosphate (Ca-TMP) for periodontal tissue engineering. Poly(ester urea) (PEU) (5% w/v) solutions containing Ca-TMP (15%, 30%, 45% w/w) were electrospun into fibrous scaffolds. The fibers were evaluated using SEM, EDS, TGA, FTIR, XRD, and mechanical tests. Degradation rate, swelling ratio, and calcium release were also evaluated. Cell/Ca-TMP and cell/scaffold interaction were assessed using stem cells from human exfoliated deciduous teeth (SHEDs) for cell viability, adhesion, and alkaline phosphatase (ALP) activity. Analysis of variance (ANOVA) and post-hoc tests were used (α = 0.05). The PEU and PEU/Ca-TMP-based membranes presented fiber diameters at 469 nm and 414-672 nm, respectively. Chemical characterization attested to the Ca-TMP incorporation into the fibers. Adding Ca-TMP led to higher degradation stability and lower dimensional variation than the pure PEU fibers; however, similar mechanical characteristics were observed. Minimal calcium was released after 21 days of incubation in a lipase-enriched solution. Ca-TMP extracts enhanced cell viability and ALP activity, although no differences were found between the scaffold groups. Overall, Ca-TMP was effectively incorporated into the PEU fibers without compromising the morphological properties but did not promote significant cell function.

Nanoscale ß-TCP-Laden GelMA/PCL Composite Membrane for Guided Bone Regeneration.

Major advances in the field of periodontal tissue engineering have favored the fabrication of biodegradable membranes with tunable physical and biological properties for guided bone regeneration (GBR). Herein, we engineered innovative nanoscale beta-tricalcium phosphate (ß-TCP)-laden gelatin methacryloyl/polycaprolactone (GelMA/PCL-TCP) photocrosslinkable composite fibrous membranes via electrospinning. Chemo-morphological findings showed that the composite microfibers had a uniform porous network and ß-TCP particles successfully integrated within the fibers. Compared with pure PCL and GelMA/PCL, GelMA/PCL-TCP membranes led to increased cell attachment, proliferation, mineralization, and osteogenic gene expression in alveolar bone-derived mesenchymal stem cells (aBMSCs). Moreover, our GelMA/PCL-TCP membrane was able to promote robust bone regeneration in rat calvarial critical-size defects, showing remarkable osteogenesis compared to PCL and GelMA/PCL groups. Altogether, the GelMA/PCL-TCP composite fibrous membrane promoted osteogenic differentiation of aBMSCs in vitro and pronounced bone formation in vivo. Our data confirmed that the electrospun GelMA/PCL-TCP composite has a strong potential as a promising membrane for guided bone regeneration.

Multifunctional and biodegradable methacrylated gelatin/Aloe vera nanofibers for endodontic disinfection and immunomodulation.

Currently employed approaches and materials used for vital pulp therapies (VPTs) and regenerative endodontic procedures (REPs) lack the efficacy to predictably achieve successful outcomes due to their inability to achieve adequate disinfection and/or lack of desired immune modulatory effects. Natural polymers and medicinal herbs are biocompatible, biodegradable, and present several therapeutic benefits and immune-modulatory properties; thus, standing out as a clinically viable approach capable of establishing a conducive environment devoid of bacteria and inflammation to support continued root development, dentinal bridge formation, and dental pulp tissue regeneration. However, the low stability and poor mechanical properties of the natural compounds have limited their application as potential biomaterials for endodontic procedures. In this study, Aloe vera (AV), as a natural antimicrobial and anti-inflammatory agent, was incorporated into photocrosslinkable Gelatin methacrylate (GelMA) nanofibers with the purpose of developing a highly biocompatible biomaterial capable of eradicating endodontic infection and modulating inflammation. Stable GelMA/AV nanofibers with optimal properties were obtained at the ratio of (70:30) by electrospinning. In addition to the pronounced antibacterial effect against Enterococcus faecalis, the GelMA/AV (70:30) nanofibers also exhibited a sustained antibacterial activity over 14 days and significant biofilm reduction with minimal cytotoxicity, as well as anti-inflammatory properties and immunomodulatory effects favoring healing. Our results indicate that the novel GelMA/AV (70:30) nanofibers hold great potential as a biomaterial strategy for endodontic infection eradication and enhanced healing.

Next-generation biomaterials for dental pulp tissue immunomodulation.

OBJECTIVES: The current standard for treating irreversibly damaged dental pulp is root canal therapy, which involves complete removal and debridement of the pulp space and filling with an inert biomaterial. A regenerative approach to treating diseased dental pulp may allow for complete healing of the native tooth structure and enhance the long-term outcome of once-necrotic teeth. The aim of this paper is, therefore, to highlight the current state of dental pulp tissue engineering and immunomodulatory biomaterials properties, identifying exciting opportunities for their synergy in developing next-generation biomaterials-driven technologies. METHODS: An overview of the inflammatory process focusing on immune responses of the dental pulp, followed by periapical and periodontal tissue inflammation are elaborated. Then, the most recent advances in treating infection-induced inflammatory oral diseases, focusing on biocompatible materials with immunomodulatory properties are discussed. Of note, we highlight some of the most used modifications in biomaterials' surface, or content/drug incorporation focused on immunomodulation based on an extensive literature search over the last decade. RESULTS: We provide the readers with a critical summary of recent advances in immunomodulation related to pulpal, periapical, and periodontal diseases while bringing light to tissue engineering strategies focusing on healing and regenerating multiple tissue types. SIGNIFICANCE: Significant advances have been made in developing biomaterials that take advantage of the host's immune system to guide a specific regenerative outcome. Biomaterials that efficiently and predictably modulate cells in the dental pulp complex hold significant clinical promise for improving standards of care compared to endodontic root canal therapy.

Composite Graded Melt Electrowritten Scaffolds for Regeneration of the Periodontal Ligament-to-Bone Interface.

Periodontitis is a ubiquitous chronic inflammatory, bacteria-triggered oral disease affecting the adult population. If left untreated, periodontitis can lead to severe tissue destruction, eventually resulting in tooth loss. Despite previous efforts in clinically managing the disease, therapeutic strategies are still lacking. Herein, melt electrowriting (MEW) is utilized to develop a compositionally and structurally tailored graded scaffold for regeneration of the periodontal ligament-to-bone interface. The composite scaffolds, consisting of fibers of polycaprolactone (PCL) and fibers of PCL-containing magnesium phosphate (MgP) were fabricated using MEW. To maximize the bond between bone (MgP) and ligament (PCL) regions, we evaluated two different fiber architectures in the interface area. These were a crosshatch pattern at a 0/90° angle and a random pattern. MgP fibrous scaffolds were able to promote in vitro bone formation even in culture media devoid of osteogenic supplements. Mechanical properties after MgP incorporation resulted in an increase of the elastic modulus and yield stress of the scaffolds, and fiber orientation in the interfacial zone affected the interfacial toughness. Composite graded MEW scaffolds enhanced bone fill when they were implanted in an in vivo periodontal fenestration defect model in rats. The presence of an interfacial zone allows coordinated regeneration of multitissues, as indicated by higher expression of bone, ligament, and cementoblastic markers compared to empty defects. Collectively, MEW-fabricated scaffolds having compositionally and structurally tailored zones exhibit a good mimicry of the periodontal complex, with excellent regenerative capacity and great potential as a defect-specific treatment strategy.

Electrospun Azithromycin-Laden Gelatin Methacryloyl Fibers for Endodontic Infection Control.

This study was aimed at engineering photocrosslinkable azithromycin (AZ)-laden gelatin methacryloyl fibers via electrospinning to serve as a localized and biodegradable drug delivery system for endodontic infection control. AZ at three distinct amounts was mixed with solubilized gelatin methacryloyl and the photoinitiator to obtain the following fibers: GelMA+5%AZ, GelMA+10%AZ, and GelMA+15%AZ. Fiber morphology, diameter, AZ incorporation, mechanical properties, degradation profile, and antimicrobial action against Aggregatibacter actinomycetemcomitans and Actinomyces naeslundii were also studied. In vitro compatibility with human-derived dental pulp stem cells and inflammatory response in vivo using a subcutaneous rat model were also determined. A bead-free fibrous microstructure with interconnected pores was observed for all groups. GelMA and GelMA+10%AZ had the highest fiber diameter means. The tensile strength of the GelMA-based fibers was reduced upon AZ addition. A similar pattern was observed for the degradation profile in vitro. GelMA+15%AZ fibers led to the highest bacterial inhibition. The presence of AZ, regardless of the concentration, did not pose significant toxicity. In vivo findings indicated higher blood vessel formation, mild inflammation, and mature and thick well-oriented collagen fibers interweaving with the engineered fibers. Altogether, AZ-laden photocrosslinkable GelMA fibers had adequate mechanical and degradation properties, with 15%AZ displaying significant antimicrobial activity without compromising biocompatibility.

Effect of sodium hexametaphosphate and quercetin, associated or not with fluoride, on dentin erosion in vitro.

OBJECTIVE: to investigate the ability of solutions containing sodium hexametaphosphate, fluoride and quercetin, alone or in association, to prevent dentin erosion and to inhibit matrix metalloproteinases -2 and -9 activity using in vitro protocols. DESIGN: Root dentin blocks (n = 96) were prepared and divided into 8 experimental groups (n = 12/group), according to the solutions to be tested: Placebo; 0.24% sodium fluoride (F); 1.0% sodium hexametaphosphate (HMP); 0.03% quercetin (QC); F+HMP; F+QC; HMP+QC; and F+HMP+QC. Erosive challenges were performed 4×/day for 5 days. Specimens were treated with the respective solutions for one minute, twice a day. Next, dentin loss (profilometry) and integrated hardness area in depth (KHN × µm) were determined. The antiproteolytic potential was assessed by gelatin zymography. Dentin erosion results (log10-transformed) were submitted to one-way ANOVA, followed by Tukey's test. Integrated hardness area in depth data (raw) were submitted to two-way, repeated-measures ANOVA, followed by Holm-Sidak's test (p<0.05). RESULTS: Dentin erosion was significantly lower for F+HMP+QC than for all other treatments. At the shallowest depths (5-30 µm), blocks treated with F+HMP+QC had the highest integrated hardness area in depth values. All treatments completely inhibited matrix metalloproteinases-2 activity, except for the group QC (77% inhibition). For matrix metalloproteinases-9, all HMP-containing solutions or F+QC promoted total antiproteolytic activity. CONCLUSION: The association of fluoride, sodium hexametaphosphate, and quercetin must be considered a valuable strategy for novel product formulation for home and professional use, considering its superior protective effects against dentin erosion and its antiproteolytic potential.

Effect of red wine or its polyphenols on induced apical periodontitis in rats.

AIM: To evaluate the effect of red wine consumption or its polyphenols on the inflammation/resorption processes associated with apical periodontitis in rats. METHODOLOGY: Thirty-two three-month-old Wistar rats had apical periodontitis induced in four first molars and were then arranged into four groups: control (C)-rats with apical periodontitis; wine (W)-rats with apical periodontitis receiving 4.28 ml/kg of red wine; resveratrol+quercetin (R+Q)-rats with apical periodontitis receiving 4.28 ml/kg of a solution containing 1.00 mg/L of quercetin and 0.86 mg/L of resveratrol and alcohol (ALC)-rats with apical periodontitis receiving the alcoholic dose contained in the wine. The oral gavage treatments were administered daily, from day 0 to day 45. On the 15th day, apical periodontitis was induced, and on the 45th day, the animals were euthanized. Histological, immunohistochemical (RANKL, OPG, TRAP, IL-10, TNF-⍺ and IL-1ß) and micro-computed tomography for bone resorption analysis were performed in the jaws. The Kruskal-Wallis with Dunn's test was performed for nonparametric data, and the anova with Tukey's test for parametric data, p < .05. RESULTS: The median score of the inflammatory process was significantly lower in the R+Q group (1) compared to the C (2) (p = .0305) and ALC (3) (p = .0003) groups, and not different from the W (1.5) group. The immunolabeling for OPG was significantly higher in the R+Q group (p = .0054) compared to all groups; the same was observed for IL-10 (p = .0185), different from groups C and ALC. The R+Q group had the lowest TRAP cell count (p < .0001), followed by the W group, both inferior to C and ALC groups. The lowest bone resorption value was in the R+Q group (0.50mm3  ± 0.21mm3 ), significantly lower (p = .0292) than the C group (0.88mm3  ± 0.10mm3 ). The W group (0.60 mm3  ± 0.25 mm3 ) and R+Q group had less bone resorption compared to the ALC group (0.97 mm3  ± 0.22 mm3 ), p = .0297 and p = .0042, respectively. CONCLUSION: Red wine administration to rats for 15 days before induction of apical periodontitis decreased inflammation, TRAP marking and periapical bone resorption compared to alcohol. Resveratrol-quercetin administration reduced the inflammatory process in apical periodontitis, periapical bone resorption, and altered the OPG, IL-10 and TRAP expression compared to C and ALC groups.

Excessive caffeine intake increases bone resorption associated with periapical periodontitis in rats.

AIM: To evaluate the effect of excessive caffeine intake on the inflammation/resorption processes associated with periapical periodontitis (PP) in rats. METHODOLOGY: Sixteen Wistar rats were used. Periapical periodontitis was induced in the four first molars in each animal. The animals were arranged into two groups: control (C)-rats with periapical periodontitis; and caffeine (CAF)-rats with periapical periodontitis under caffeine administration protocol. The CAF animals received 10 mg/100 g of body weight/day of caffeine via gavage starting fifteen days before PP induction and continuing for thirty more days until euthanasia. On the 30th day, the animals were euthanized and the jaws removed for microcomputed tomography, histological and immunohistochemical analysis for RANKL, OPG, TRAP, IL-10, TNF-⍺ and IL-1ß. The Mann-Whitney test was performed for nonparametric data, and Student's t test was performed for parametric data, using p < .05. RESULTS: There was no significant difference in the weight change between the groups. The median score of the inflammatory process was significantly greater in the CAF group (3) compared with the C group (2), p = .0256. Bone resorption was greater in the group consuming caffeine (1.08 ± 0.15 mm3 ) compared with the C group (0.88 ± 0.10 mm3 ), p = .0346. The immunolabelling for RANKL, TRAP and IL-1ß was significantly higher in the CAF group when compared to the control, p < .05. No differences were found for the OPG, IL-10 and TNF-⍺ immunolabelling. CONCLUSION: Excessive caffeine exposure via gavage in rats was able to exacerbate the volume of periapical bone destruction, and the inflammatory pattern deriving from periapical periodontitis altering the expression of RANKL, IL-1ß and TRAP.

Dietary supplementation with multi-strain formula of probiotics modulates inflammatory and immunological markers in apical periodontitis.

OBJECTIVE: The aim of this study was to evaluate whether probiotics multi-strain formula affects the development of apical periodontitis (AP) induced in rats. METHODOLOGY: 16 Wistar rats were divided in two groups (n=8): rats with AP fed with regular diet (Control-C (CG)); rats with AP, fed with regular diet and supplemented with multi-strain formula (one billion colony-forming units (CFU)): GNC Probiotic Complex (PCG) ( Lactobacillus acidophilus, Lactobacillus salivaris, Lactobacillus plantarum, Lactobacillus rhamnosus, Bifidobacterium bifidum, Bifidobacterium animalis subs. lactis and Streptococcus thermofilus ). AP was induced in the upper and lower first molars by dental pulp exposure to the oral environment. PCG was administered orally through gavage for 30 days during the AP development. After this period the animals were euthanized and the mandibles were removed and processed for histologic analysis, and immunochemical assays for interleukin (IL)-6, IL-10, IL-1ß, RANKL, OPG, and TRAP. The Mann-Whitney U test and Student's t test were performed (P<.05). RESULTS: The CG showed more intense inflammatory infiltrate than the PCG group (P<.05). IL-1ß, IL 6 and RANKL decreased in the PCG group compared with CG (P<.05). The IL-10 level increased in the PCG group (P<.05). The OPG level was similar in both groups (P>.05). The number of mature osteoclasts (TRAP-positive multinucleated cells) was lower in PCG group when compared to the CG (P<.05). CONCLUSION: Probiotic Complex modulates inflammation and bone resorption in apical periodontitis.

Influence of the Vehicle on the Tissue Reaction and Biomineralization of Fast Endodontic Cement

ABSTRACT Objective: To investigate the tissue response and the biomineralization ability of CER prepared with epoxy resin or water compared to Mineral Trioxide Aggregate (MTA). Material and Methods: Polyethylene tubes containing materials or empty tubes for control were inserted into the subcutaneous tissues of 30 rats. After 7, 15, 30, 60, and 90 days, the rats were killed and the tubes were removed for analysis using hematoxylin-eosin staining, von Kossa staining, and under polarized light. Inflammation was graded through a score system; the thickness of the fibrous capsule was classified as thin or thick; the biomineralization ability was recorded as present or absent. The results were statistically analyzed using the Kruskal-Wallis test (p<0.05). Results: Histologic analysis performed after 7 and 15 days for CER prepared with epoxy resin or water and for MTA showed moderate inflammation and a thick fibrous capsule (p>0.05). After 30, 60, and 90 days, mild inflammation, and a thin fibrous capsule were observed in all groups (p>0.05). Conclusion: All materials had structures positive for von Kossa and birefringent to polarized light. CER epoxy resin showed biocompatibility and biomineralization similar to CER water and MTA.

Dietary supplementation with multi-strain formula of probiotics modulates inflammatory and immunological markers in apical periodontitis

Abstract Objective The aim of this study was to evaluate whether probiotics multi-strain formula affects the development of apical periodontitis (AP) induced in rats. Methodology 16 Wistar rats were divided in two groups (n=8): rats with AP fed with regular diet (Control-C (CG)); rats with AP, fed with regular diet and supplemented with multi-strain formula (one billion colony-forming units (CFU)): GNC Probiotic Complex (PCG) ( Lactobacillus acidophilus, Lactobacillus salivaris, Lactobacillus plantarum, Lactobacillus rhamnosus, Bifidobacterium bifidum, Bifidobacterium animalis subs. lactis and Streptococcus thermofilus ). AP was induced in the upper and lower first molars by dental pulp exposure to the oral environment. PCG was administered orally through gavage for 30 days during the AP development. After this period the animals were euthanized and the mandibles were removed and processed for histologic analysis, and immunochemical assays for interleukin (IL)-6, IL-10, IL-1β, RANKL, OPG, and TRAP. The Mann-Whitney U test and Student's t test were performed (P<.05). Results The CG showed more intense inflammatory infiltrate than the PCG group (P<.05). IL-1β, IL 6 and RANKL decreased in the PCG group compared with CG (P<.05). The IL-10 level increased in the PCG group (P<.05). The OPG level was similar in both groups (P>.05). The number of mature osteoclasts (TRAP-positive multinucleated cells) was lower in PCG group when compared to the CG (P<.05). Conclusion Probiotic Complex modulates inflammation and bone resorption in apical periodontitis.

Cytotoxicity, inflammation, biomineralization, and immunoexpression of IL-1ß and TNF-α promoted by a new bioceramic cement.

Aim To evaluate the cytotoxicity, biocompatibility and mineralization capacity of BIO-C PULPO, and MTA. Methodology L929 fibroblasts were cultured and MTT assay was used to determine the material cytotoxicity on 6, 24, and 48 h. A total of 30 male rats (Wistar) aged between 4 and 6 months, weighing between 250 and 300 g were used. Polyethylene tubes containing BIO-C PULPO, MTA, and empty tubes were implanted into dorsal connective tissue. After the experimental periods (7, 15, 30, 60, and 90 days) the tubes were histologically analyzed using hematoxylin-eosin (H&E), immunolabeling of IL-1ß and TNF-α, and von Kossa staining, or without staining for polarized light analysis. The average number of inflammatory cells was quantified; the mineralization assessment was determined by the area marked in µm2 and semiquantitative immunolabeling analyses of IL-1ß and TNF-α were performed. Then, data underwent statistical analysis with a 5% significance level. Results It was observed that BIO-C PULPO and MTA presented cytocompatibility at 6, 24, and 48 similar or higher than control for all evaluated period. On periods 7 and 15 days, BIO-C PULPO was the material with the highest number of inflammatory cells (p<0.05). On periods 30, 60, and 90 days, BIO-C PULPO and MTA presented similar inflammatory reactions (p>0.05). No statistical differences were found between Control, BIO-C PULPO, and MTA for immunolabeling of IL-1ß and TNF-α in the different periods of analysis (p<0.05). Positive von Kossa staining and birefringent structures under polarized light were observed in all analyzed periods in contact with both materials, but larger mineralization area was found with BIO-C PULPO on day 90 (p<0.05). Conclusion BIO-C PULPO was biocompatible and induced mineralization similar to MTA.

Cytotoxicity, inflammation, biomineralization, and immunoexpression of IL-1β and TNF-α promoted by a new bioceramic cement

Abstract Aim To evaluate the cytotoxicity, biocompatibility and mineralization capacity of BIO-C PULPO, and MTA. Methodology L929 fibroblasts were cultured and MTT assay was used to determine the material cytotoxicity on 6, 24, and 48 h. A total of 30 male rats (Wistar) aged between 4 and 6 months, weighing between 250 and 300 g were used. Polyethylene tubes containing BIO-C PULPO, MTA, and empty tubes were implanted into dorsal connective tissue. After the experimental periods (7, 15, 30, 60, and 90 days) the tubes were histologically analyzed using hematoxylin-eosin (H&E), immunolabeling of IL-1β and TNF-α, and von Kossa staining, or without staining for polarized light analysis. The average number of inflammatory cells was quantified; the mineralization assessment was determined by the area marked in μm2 and semiquantitative immunolabeling analyses of IL-1β and TNF-α were performed. Then, data underwent statistical analysis with a 5% significance level. Results It was observed that BIO-C PULPO and MTA presented cytocompatibility at 6, 24, and 48 similar or higher than control for all evaluated period. On periods 7 and 15 days, BIO-C PULPO was the material with the highest number of inflammatory cells (p<0.05). On periods 30, 60, and 90 days, BIO-C PULPO and MTA presented similar inflammatory reactions (p>0.05). No statistical differences were found between Control, BIO-C PULPO, and MTA for immunolabeling of IL-1β and TNF-α in the different periods of analysis (p<0.05). Positive von Kossa staining and birefringent structures under polarized light were observed in all analyzed periods in contact with both materials, but larger mineralization area was found with BIO-C PULPO on day 90 (p<0.05). Conclusion BIO-C PULPO was biocompatible and induced mineralization similar to MTA.

Effects of different alcohol concentrations on the development of apical periodontitis in rats.

AIM: To investigate the effect of different alcohol concentrations on the development of apical periodontitis (AP) in rats. METHODS: Forty Wistar rats were arranged into five groups: (C) - control rats receiving sterile water as the only liquid; (G5) - animals receiving an alcohol solution at 5%, (G10) - alcohol solution at 10%, (G15) - alcohol solution at 15%, and (G20) - alcohol solution at 20%. The alcoholic solution or water was given to the groups as the sole source of hydration throughout the 30 days of the experiment. AP was induced in the mandibular molars on the first day. In the end, the animals were euthanized for histopathological and IL-1b, RANKL, OPG, and TRAP analyses. The Kruskal-Wallis test was used for nonparametric data, and ANOVA followed by the Tukey test were performed for parametric data, p < 0.05. RESULTS: G15 and G20 had a greater chronic inflammatory infiltrate (Score 3) and AP size bigger (1.59 ±â€¯0.41 and 1.83 ±â€¯0.38, respectively) than the C, G5 and G10 (p < 0.05). No significant difference was found in the IL-1b analyses. The G15 and G20 showed the highest immunolabeling pattern for RANKL and the lowest for OPG. The G20 had greater TRAP cells per mm (4.70 ±â€¯0.99) compared to the C, G5, and G10 (p < 0.05). Furthermore, G15 presented 3.92 ±â€¯0.64 TRAP cells/mm, higher than C (p < 0.05). CONCLUSIONS: G5 and G10 did not exert a protective or aggravating effect on the AP development. However, G15 and G20 had a significant effect on the AP severity, exacerbating the inflammation and osteoclast markers.

Hypertension affects the biocompatibility and biomineralization of MTA, High-plasticity MTA, and Biodentine®.

This study evaluated the effect of hypertension on tissue response and biomineralization capacity of white Mineral Trioxide Aggregate (MTA), High-plasticity MTA (MTA HP), and Biodentine® (BDT) in rats. Polyethylene tubes filled with MTA, MTA HP, BDT, and the control group (empty tubes) were placed into the dorsal subcutaneous tissue of 32 male rats (16 normotensive (NT) and 16 hypertensive rats - 8 per group). After 7 and 30 days, the polyethylene tubes surrounded by connective tissue were removed, fixed, and embedded in histological resin. The mean number of inflammatory cells was estimated in HE-stained sections, biomineralization was quantified as area (µm2) by Kossa (VK) staining, and examination by polarized light (LP) microscopy was performed. The differences amongst the groups were analyzed statistically by the Mann-Whitney or Student's t test, according to Shapiro-Wilk test of normality (p < 0.05). The inflammatory responses to all materials were greater in hypertensive rats than in NT rats (p < 0.05). Positive VK staining in MTA and BDT were more pronounced in NT rats at 7 and 30 days (p < 0.05). Birefringent structures in LP for MTA, MTA HP, and BDT were more pronounced in NT rats at 7 days (p<0.05). In rats, hypertension was able to increase inflammatory infiltrate and decrease biomineralization of the tested materials.

Chronic alcohol consumption changes blood marker profile and bone density in rats with apical periodontitis.

AIM: The aim of the present study was to evaluate apical periodontitis (AP) development in rats under a chronic alcohol diet by calcium, phosphorus, and alkaline phosphatase blood levels in addition to histological and radiographic analyses. METHODS: Thirty-two rats were arranged into four groups: (a) group 1: without apical periodontitis and on a regular diet; (b) group 2: AP and on a regular diet; (c) group 3: alcoholic diet without apical periodontitis; and (d) group 4: alcoholic diet and apical periodontitis. Alcoholic solution at 20% was given throughout the 8-week experiment. AP was induced in the first molars at the end of the 7th week. At the end, the animals were anesthetized for blood collection, followed by euthanasia, and jaws were removed for digital radiography and histological processing. The level of significance was 5%. RESULTS: Calcium levels remained constant in all groups (P > 0.05). Group 4 showed a higher phosphorous level than group 2 (P < 0.05). The alkaline phosphatase activity was higher in group 3 compared with group 1 (P < 0.05). Three animals in group 4 exhibited a severe inflammatory reaction, whereas the animals in group 2 did not demonstrate any reaction (P < 0.05). The lowest value of radiographic density was given by group 4 (P < 0.05). CONCLUSIONS: Chronic alcohol consumption increased serum phosphorus and decreased bone density in the periapical region, favoring AP development.